PROJECT 3: KOO/ROY - ABSTRACT The amyloid precursor protein (APP) is sequentially cleaved by - and -secretases to generate amyloid -peptide (A) in the brain - a central player in the amyloid cascade hypothesis. APP cleavage by -secretase-1 (BACE-1) is the rate-limiting step for production of A. A is believed to exert its toxicity on neurons while in a soluble and oligomeric state, prior to deposition as insoluble fibrils in brain. Thus, for reasons related to both pathophysiology and therapeutics, understanding mechanisms and pathways of A generation from APP is a major focus of many laboratories. An intriguing aspect of A production is that its release is dependent upon neuronal activity - enhanced synaptic activity results in more A release. Though pathways involved in trafficking and cleavage of APP in neurons are of obvious importance, the vast majority of previous studies on APP/BACE-1 trafficking have been carried out in non-neuronal cells. These findings may not always be applicable to neurons, which are highly polarized and are known to have very different trafficking mechanisms. Furthermore, inferences on how neuronal activity modulates APP processing by BACE-1 require work in neurons. The prevailing view is that at presynaptic terminals, heightened synaptic vesicle recycling that accompanies high synaptic activity results in increased internalization into endosomes of APP where proteolysis by secretases take place. However, our recent studies using live neuronal imaging showed rather surprising results in that APP and BACE-1 normally traffic in distinct vesicles - perhaps preventing unabated cleavage - but converge in dendrites upon activity-induction. This led us to propose a new model whereby neuronal activity brings together APP and BACE-1 in dendrites where the two molecules interact. Only subsequently are these two molecules sorted into axons to distal terminals. Accordingly, this Project will examine a number of predictions that emanate from this working model and will allow us to refine the trafficking pathways of APP and BACE-1 and define how they relate to amyloidogenesis in neurons in an activity-dependent manner. Four Aims are proposed: 1) test the hypothesis that APP and BACE-1 are first sorted to dendrites immediately after biosynthesis, 2) determine the subcellular sites where APP and BACE-1 interact and correlate this to sites of A release, 3) test the hypothesis that APP and BACE-1 are transcytosed from dendrites to axons, possibly in an activity dependent manner, and 4) utilize tissue from the Neuro-pathology Core to assess whether the colocalization of APP and BACE-1 is preferential to the default mode network brain regions which are known to be sites of A deposits. Collectively, results from these studies will provide new insights into the trafficking pathways of APP and BACE-1 and demonstrate how neuronal activity modulates these pathways to enhance APP cleavage and A release.